In the proposed experiments, we will determine the potential of two series of chemical carcinogens for producing nucleotide damage in DNA in vitro. As a sensitive test system we will utilize phiX174 Replicative Form (RF) I DNA, a closed circular, supercoiled, doublestranded DNA of MW 3.4 x 10 to the 6th power. With this DNA, as little as 1 in 10 to the 5th power altered nucleotides can be easily detected under neutral conditions; this amounts to 1 per 10 molecules. The carcinogens to be used are: 1) acetylaminofluorene (AFF) and its derivatives, N-hydroxyacetylaminofluorene (HO-AAF) and N-acetoxy-acetylaminofluorene(AcO-AAF) and 2) benz(a)anthracene (BA) and dibenz(a,h) anthracene (DBA) and several dihydrodiol and epoxide derivatives. Particular attention will be paid to the suspected "ultimate" cardinogenic forms of these, AcO-AAF and the K-region epoxides of BA and DBA. Following reaction of the chemical with the RFI DNA at physiological pH, the unbound carcinogen will be removed by column chromatography and the RF DNA directly assayed for nucleotide damage and strand breaks by sucrose gradient sedimentation, or further treated by alkali, near UV light or exposure to extracts of human embryonic lung cells, followed by sucrose gradient sedimentation. The gradients will be fractionated, the labeled peaks located and quantitated by liquid scintillation counting and the biological activity of the fractions assayed by the ability of the RF DNA to infect host bacterial spheroplasts. Spheroplasts from a series of well defined radiation repair mutants will be used to determine the ability of specific repair systems to act on carcinogen treated DNA, and to detect the presence of unrepairable nucleotide damage which may be responsible for the carcinogenesis of normal cells.